Bottom Line:
Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C.We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli.Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+).

ABSTRACTAquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na(+) for energy coupling of the flagellar motor. The Na(+)-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.

f3: Swimming speed of A. aeolicus in solution.(A) Photograph of the variable-temperature chamber used in this study. (B) Histogram of swimming speed of the cells at 85 °C, the optimal growth temperature. (C) Thermo-dependent swimming speed of the A. aeolicus cells. The cells were grown at 85 °C and observed at each temperature.

Mentions:
Next, we examined whether A. aeolicus could swim in solution. We enclosed culture broth to a variable–temperature chamber for optical microscopy (Fig. 3A), and then monitored the cells at different temperatures. At a temperature of 85 °C, we observed that only a low population of cells swam smoothly (Movie S1), while most of the cells did not show motility, consistent with the TEM observation result that most cells did not have flagellar filaments. The swimming speed of the cells was 93 ± 40 μm s−1 (mean ± SD, n = 55) at 85 °C (Fig. 3B,C). Most of the cells swam linearly, however, a few cells showed changes in direction during their swimming. The swimming speed decreased with decreasing temperature of the chamber, and exhibited 16 ± 5 μm s−1 (mean ± SD, n = 90) at room temperature (22 °C) (Fig. 3C). These results indicate that the motility machinery, presumably the flagellum, of A. aeolicus functions well at high-temperature conditions and needs high-temperature for its maximum function.

f3: Swimming speed of A. aeolicus in solution.(A) Photograph of the variable-temperature chamber used in this study. (B) Histogram of swimming speed of the cells at 85 °C, the optimal growth temperature. (C) Thermo-dependent swimming speed of the A. aeolicus cells. The cells were grown at 85 °C and observed at each temperature.

Mentions:
Next, we examined whether A. aeolicus could swim in solution. We enclosed culture broth to a variable–temperature chamber for optical microscopy (Fig. 3A), and then monitored the cells at different temperatures. At a temperature of 85 °C, we observed that only a low population of cells swam smoothly (Movie S1), while most of the cells did not show motility, consistent with the TEM observation result that most cells did not have flagellar filaments. The swimming speed of the cells was 93 ± 40 μm s−1 (mean ± SD, n = 55) at 85 °C (Fig. 3B,C). Most of the cells swam linearly, however, a few cells showed changes in direction during their swimming. The swimming speed decreased with decreasing temperature of the chamber, and exhibited 16 ± 5 μm s−1 (mean ± SD, n = 90) at room temperature (22 °C) (Fig. 3C). These results indicate that the motility machinery, presumably the flagellum, of A. aeolicus functions well at high-temperature conditions and needs high-temperature for its maximum function.

Bottom Line:
Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C.We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli.Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+).

ABSTRACTAquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na(+) for energy coupling of the flagellar motor. The Na(+)-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.